A novel cysteine fluorescent probe based on benzothiazole and quinoline with a large stokes shift and application in living cell and mice

Article abstract of DOI:10.1016/j.jphotochem.2021.113335

A novel fluorescent probe based on hydroxyquinoline conjugated with benzothiazolyl unit dye Doc-Ac was prepared. The results of fluorescent and absorption spectroscopic responses towards amino acids showed that the probe had a capability of rapidly and selectively detecting cysteine over other amino acids with a large stokes shift in aqueous solution. The sensing mechanism was proved by the proton NMR and high resolution mass spectrometry analysis. The fluorescence images indicated that this probe could discriminate the exogenous/endogenous cysteine in living cells and mice.

Full text of DOI:10.1016/j.jphotochem.2021.113335

Journal of Photochemistry & Photobiology, A: Chemistry 418 (2021) 113335
Contents lists available at ScienceDirect
Journal of Photochemistry & Photobiology, A: Chemistry
journal homepage: www.elsevier.com/locate/jphotochem
A novel cysteine fluorescent probe based on benzothiazole and quinoline
with a large stokes shift and application in living cell and mice
Huihui Mei, Xin Gu, Minghui Wang, Yonghong Cai*, Kuoxi Xu*
Henan Engineering Research Center of Industrial Recirculating Water Treatment, College of Chemistry and Chemical Engineering, Henan University, 475004, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords:
A novel fluorescent probe based on hydroxyquinoline conjugated with benzothiazolyl unit dye Doc-Ac was
prepared. The results of fluorescent and absorption spectroscopic responses towards amino acids showed that the
probe had a capability of rapidly and selectively detecting cysteine over other amino acids with a large stokes
shift in aqueous solution. The sensing mechanism was proved by the proton NMR and high resolution mass
spectrometry analysis. The fluorescence images indicated that this probe could discriminate the exogenous/
endogenous cysteine in living cells and mice.
Cysteine
Fluorescent probe
Rapid response
Large stokes shift
Cells and mice imaging
1. Introduction
more attention [27–31]. It is known that fluorescent probe with larger
Stokes shift (more than 80 nm) [32–34] is more suitable for bioimaging
Cysteine (Cys), as a product of homocysteine (Hcy) metabolize and a
precursor of the antioxidant glutathione (GSH) as a potential biomarker
for various diseases, is attracting an increasing attention in various
applications in vivo [35–37] because it can improve the detection
sensitivity by minimizing self-absorption and auto-fluorescence caused
by the minimal the overlap between the excitation and fluorescence
biological systems [1,2]. The abnormal levels of Cys (30–200
μM) [3] in
spectra ordinarily results in a poor signal-to-noise ratio and
the body are closely associated with a variety of diseases [4–6], such as
Parkinson’s disease, Alzheimer’s disease, cardiovascular disease, cancer,
liver damage, slow growth in children, rheumatoid arthritis, neurotox-
icity, edema or skin lesions, and so on. Hence, accurately detecting of
Cys is especially important for understanding its physiological function
and clinical index in medicine. Over the past decades, several analytical
methods including high-performance liquid chromatography [7] elec-
trochemical detectable analytical techniques [8–10], mass spectrometry
detector [11] have been developed. Compared with these methods,
fluorescent probes due to the advantages of simple operation, low cost,
fast response and low detection limit have attracted increasing attention
of many researchers [12–14]. Some recently reported fluorescent probes
showed highly detection capacity for Cys (Table S1). Such as some
amino acid fluorescent probes based on the sulfhydryl sensing mecha-
nisms have been reported disulfide or thioether or thioester [15,16],
cyclization with aldehydes [17,18], sulfonate [19,20], conjugated
addition-cyclization with acrylates [21,22], cleavage reactions [23],
nucleophilic substitution [24], michael addition [25] and others [26].
Promoted by the conjugated addition-cyclization reaction with acry-
lates, fluorescent probes for the detection of Cys attracted more and
self-quenching originated from by backscattering from biological sam-
ples. Nevertheless, some reported fluorescent probes showed highly
detection capacity for the detection of Cys. However, most of them
require high content organic solvents or have relatively small Stoke
shifts which make them unsuitable for biological applications. There-
fore, there is an urgent need to develop a probe that can simultaneously
satisfy a large Stoke shift and good water solubility.
Based on our previous work [38–41], we developed a novel fluo-
rescent probe, Doc-Ac, utilizing hydroxyquinoline conjugated with a
charged benzothiazolyl unit though unsaturated olefinic bond as fluo-
rophore which also featured high water solubility and high reactivity of
the phenolic hydroxyl group, The acryloyl group is introduced as a
recognition part by removing a hydrogen atom and a chlorine atom
(Scheme 1). The probe displayed highly selectivity to Cys over Hcy and
GSH with a large Stokes shift (165 nm) and fast fluorescence response
(within 7 min.) in almost 100 % aqueous solution (comprising 0.3 %
dimethyl sulfoxide, DMSO) at a physiological pH. In addition, the probe
displayed low toxicity and successfully used in fluorescence imaging of
endogenous Cys in living cells and mice. As far as we know, there are
very few reports of real-time visualization and rapid response of Cys in a
* Corresponding authors at: Henan Engineering Research Center of Industrial Recirculating Water Treatment, College of Chemistry and Chemical Engineering,
Henan University, Kaifeng, Henan, 475004, PR China.
E-mail addresses: caiyonghaong@henu.edu.cn (Y. Cai), xukx@henu.edu.cn (K. Xu).
https://doi.org/10.1016/j.jphotochem.2021.113335
Received 30 January 2021; Received in revised form 10 April 2021; Accepted 29 April 2021
Available online 24 May 2021
1010-6030/© 2021 Elsevier B.V. All rights reserved.

H. Mei et al.
Journal of Photochemistry & Photobiology, A: Chemistry 418 (2021) 113335
100 % aqueous solution.
Meanwhile, the conjugated benzothiazolyl–quinoline structure could
formed a long emission wavelengths fluorescent probe platform.
Therefore, probe Doc-Ac was expected to show the nucleophilic attack of
2. Experimental
–
Cys toward the conjugated C C double bond of acryloyl group unit and
–
formation Doc-OH, which quenched the fluorescence of Doc-Ac. The
compound Doc-Ac was obtained readily in four convenient steps under
facile conditions with a high yield starting with commercially available
raw material 2-methylbenzothiazole and 8-hydroxyquinaldine (Scheme
1). The structure of target compound Doc-Ac was characterized by 1H
NMR, 13C NMR and HRMS spectrometry (Fig. S1–S3, ESI†).
2.1. General methods
All the reagents and instruments used in the experiment, details of
experimental results and spectrometric studies can be found in Sup-
porting Information.
2.2. Synthesis
3.2. Spectroscopic studies
2.2.1. Synthesis of compound 3
In a 50 mL round bottom flask, acryloyl chloride (0.43 g, 4.80 mmol)
was added to the solution of acetonitrile (10 mL) containing of ethyl-
diisopropylamine (0.61 g, 6.00 mmol) and compound 2 (0.52 g, 3.00
mmol), the reaction was carried out at 0 ◦C for 30 min. Then transferred
to room temperature for 12 h. After the reaction, the solvent was
evaporated and the product was purified by silica gel column chroma-
tography with eluent (PE/EA, v/v, 10/1). Compound 3 was obtained as
a white precipitate (0.76 g, 80 %). 1H NMR (CDCl3) δ 10.12 (s, 1 H), 8.33
(d, J =8.5 Hz, 1 H), 8.04 (d, J =8.5 Hz, 1 H), 7.88–7.77 (m, 1 H), 7.69 (t,
J =7.9 Hz, 1 H), 7.59 (d, J =7.5 Hz, 1 H), 6.85–6.70 (m, 1 H), 6.56 (dd, J
= 17.3, 10.4 Hz, 1 H), 6.14 (d, J =10.4 Hz, 1 H).
With the probe Doc-Ac in hand, we first tested its spectroscopic
changes upon the addition of Cys and other amino acids (such as Cit, Ser,
Lys, Gly, Gln, Hyd, Val, Try, His, Ile, Phe, Thr, Glu, Trp, Arg, Asn, Met,
Hcy and GSH, 10 equiv.) in Hepes buffer (10 mM, pH 7.3, 0.3 % DMSO)
solution and this system was used in all the spectroscopic experiments.
As shown in Fig. 1a, the free probe Doc-Ac (33.33 μM) displayed a
moderate fluorescence emission around 575 nm (λex =410 nm). Once
10 equiv. of Cys was added, an obvious fluorescent quench was
observed, accompanied by an obvious color changes from yellow to
wathet under 365 nm ultraviolet radiation (inset of Fig. 1a). Except Hcy
and GSH caused some fluorescent response, other competitive analytes
caused negligible changes. As contrast to Cys, fluorescence changes of
probe Doc-Ac upon addition of other common analytes (such as Na+, K+,
Ag+, Mg2+, Mn2+, Ba2+, Ca2+, Cd2+, Zn2+, Al3+, Cr3+, Fe3+, H2O2) were
almost negligible under the same conditions (Fig. S4). Subsequently, the
2.2.2. Synthesis of compound Doc-Ac
Compound 1 (0.38 g) and compound 3 (0.44 g) were added to a 50
mL round bottom flask containing of 10 mL ethanol, then the reaction
was refluxed for 12 h. After that, the solvent was vacuum concentrated
to about 10 mL, and it was placed in the refrigerator and sealed for 12 h.
The compound Doc-Ac, a red precipitate, was obtained by filtering.
(0.62 g, 76％). 1H NMR (DMSO-d6) δ 8.70 (d, J =8.5 Hz, 1 H), 8.54 (d, J
=8.1 Hz, 1 H), 8.43 (d, J =8.4 Hz, 1 H), 8.32 (t, J =10.1 Hz, 3 H), 8.27
(d, J =8.8 Hz, 1 H), 8.03 (dd, J = 7.5, 1.8 Hz, 1 H), 7.95 (t, J =7.8 Hz, 1
H), 7.75 (t, J =7.7 Hz, 2 H), 6.72 (d, J =13.8 Hz, 2 H), 6.34 (d, J =6.0 Hz,
1 H), 4.94 (q, J =7.1 Hz, 2 H), 1.53 (t, J=7.2 Hz, 3 H); 13C NMR (DMSO-
d6) δ 170.86, 164.76, 152.07, 147.60, 146.90, 141.55, 140.77, 138.37,
134.53, 130.45, 129.90, 129.51, 129.46, 128.75, 128.08, 126.60,
125.28, 123.58, 123.06, 118.43, 117.52, 56.50, 19.03. HRMS (ESI, m/z)
calcd for [C23H19N2O2S]+: 387.1162, found: 387.1163.
UV–vis spectra of Doc-Ac (33.33 μM) towards amino acids were inves-
tigated. As shown in Fig. 1b, probe Doc-Ac showed a strong bond at 377
nm. Similar with the changes of fluorescence, the absorption band was
obviously decreased with the introduction of Cys, accompanying
obvious solution color changes (inset of Fig. 1b). The thin-layer silica gel
plate experiments presented that the color changes of Cys in natural
light (Fig. 1c) and fluorescence under 365 nm ultraviolet lamp (Fig. 1d)
were different from other analytes. The results showed the probe Doc-Ac
could be used to efficiently detection of Cys from other relevant
analytes.
The titration experiments of probe Doc-Ac towards Cys were further
performed. As shown in Fig. 2a, probe Doc-Ac had a remarkable fluo-
rescent at 575 nm and a larger Stokes shift (165 nm). With addition of
Cys, the fluorescence gradually decreased. When the concentration of
Cys reached 2.20 equiv. of the probe, the fluorescence was quenched
completely. A good non-linear curve fitting between fluorescence in-
tensity at 575 nm and vs 0–2.20 equiv. of Cys was obtained with a linear
coefficient R over 0.99 (insert of Fig. 2a) from the equation. The reaction
constant (Ka) [44,45] and limit of detection (LOD) [46,47] of probe
3. Results and discussion
3.1. Design and synthesis
As shown in Scheme 1, the probe contained an electron-donating
acrylatoxy quinoline derivative and an electron-withdrawing posi-
tively-charged benzothiazolyl unit, which were connected though an
unsaturated olefinic bond formed a ‘push-pull’ conjugation dye struc-
ture bearing intramolecular charge transfer (ICT) effect [42,43].
towards Cys were calculated to be 2.24 × 103 Mꢀ 1 and 0.07
μM (Fig. S5),
respectively. These results indicated that probe Doc-Ac could be applied
for high sensitivity detection of Cys. However, only somewhat degree
Scheme 1. Synthesis of probe Doc-Ac.
2

H. Mei et al.
Journal of Photochemistry & Photobiology, A: Chemistry 418 (2021) 113335
Fig. 1. (a) Fluorescence spectrum changes of
probe Doc-Ac (33.33 μM) for common amino
acids (10 equiv.) in Hepes buffer (10 mM, pH
7.3, 0.3 % DMSO) solution, inset: color change
of probe Doc-Ac before and after adding Cys
under 365 nm ultraviolet radiation. (b) Changes
in the UV absorption spectra of Doc-Ac(33.33
μ
M) probes of common amino acids (10
equiv.) in Hepes buffer (10 mM, pH 7.3, 0.3 %
DMSO), inset: color change of probe Doc-Ac
before and after adding Cys in natural light.
The color of probe and amino acid thin-layer
silica gel plate coloring experiment under (c)
natural light, (d) 365 nm ultraviolet radiation.
fluorescent decrease were observed for the counterparts Hcy and GSH,
indicated that this probe was able to be further utilized to differentiate
Cys from Hcy and GSH in physiological conditions (Figs. S6 and S7). As
showed in Fig. 2b, the free probe Doc-Ac showed a strong absorption
peak at 377 nm and a weak peak at 290 nm. Upon the gradual increasing
of Doc-Ac, the peak at 377 nm gradually decreased and the peak at 290
nm gradually shifted to 280 nm. Meanwhile, a new peak appeared at 320
nm with two isosbestic points at 300 and 330 nm, which indicated that
the probe Doc-Ac could highly selectively determine Cys.
6.70 and 6.34 ppm. With addition of 1 equiv. of Cys, the proton signals
the acrylate group on probe Doc-Ac were shifted to higher field, indi-
cating the formation of cyclization product. A new single peak at 10.09
ppm was found corresponding to the proton signal of phenolic hydroxyl
group. Other proton signals in aliphatic region also matched well with
that of cyclization product.
3.4. Imaging experiment
As shown in Fig. 2c, the anti-interference experiments showed that
common co-existence amino acids did not cause the obvious fluorescent
variation of probe Doc-Ac for Cys detection, except for a small decrease
in fluorescence by Hcy and GSH, which suggested that probe Doc-Ac
could be used to efficiently discriminate Cys from other relevant ana-
lytes. Time-dependent fluorescence spectra changes were investigated.
Upon the addition of Cys (2.20 equiv.), the fluorescence at 575 nm
decreased and fade away within 7 min (Fig. 2d). Compared with the
fluorescence changes towards GSH and Hcy (Fig. S8), probe Doc-Ac
presented a similar responsive-time for Cys (and all the subsequent
fluorescent data were measured 7 min after the addition of analytes). As
described in Figs. 1a and 2 a, the completely quenched fluorescence at
575 nm suggested that probe Doc-Ac possessed an excellent detection
capability for Cys.
Encouraged by the excellent photophysical property (good water-
solubility, a large stokes shift and high sensitivity) in vitro, we evalu-
ated the probe Doc-Ac capability of fluorescent bioimaging in living
cells. The results of the effect of various pH values from 2.3 to 12.3 on
the probe and its response to Cys showed optimum pH condition for
detection of Cys was in the range 6.0–8.0 (Fig. S10), indicated that probe
could be used well under physiological conditions. The cytotoxicity of
probe was also evaluated by MTT assays using SHSY5Y cells. When the
cells were incubated with 50 μM probe for 24 h, its viabilities exceed 90
% (Fig. S11), indicated the probe Doc-Ac had low biotoxicity and could
be used as detection Cys under physiological condition. The laser
confocal fluorescence images experiments were carried out for the
detection of intracellular Cys. The SHSY5Y cells were non-fluorescent
when they were not pretreated with N-methyl maleimide (NEM, a
thiol-blocking reagent, 1 mM) for 30 min (Fig. 4b) and a red fluores-
cence could be observed when the cells were further incubated with
3.3. Proposed sensing mechanism
NEM and Doc-Ac (10 μM) for 30 min (Fig. 4c), indicated the probe Doc-
The above results confirmed that the acrylate group of probe Doc-Ac
could be used as an efficient detection moiety for the determination of
Cys. In order to further verify the proposed sensing mechanism showed
in Scheme 2, the high resolution mass spectrometry (HRMS) analysis
and proton NMR of Doc-Ac treated with Cys were employed. The HRMS
(Fig. S9, ESI†) showed the characteristic peaks of m/z 333.1057 and
198.0195, which were responded to the product (Doc-OH, calcd.
333.1056) and the product of cyclization (+Na+, calcd. 198.0195),
respectively. Thus, we can infer that the acrylate group can be used as a
highly selective determination unit for sensing Cys. The 1H NMR ex-
periments were performed to confirm the mechanism. As shown in
Fig. 3a, the free probe showed the acrylate group proton signals at 6.75,
Ac could permeate the cell membrane and detect intracellular Cys. The
control experiments were further performed. By contrast, the cells were
pretreated with the NEM (1 mM) for 30 min, which could decrease the
intracellular Cys, then added to Cys (100
Doc-Ac (10
μM) and further incubated with
μ
M) for another 30 min. No fluorescence was observed
(Fig. 4d). These results confirmed that the probe Doc-Ac could be
applied as a fluorescent probe for the detection of Cys in living cell.
Kunming mice (about 25 g) were used for live animal vivo imaging
experiments model for monitoring Cys. The mice were injected subcu-
taneously with saline (20 μM, left side) and probe Doc-Ac (20 μM, right
side), respectively. As illustrated in Fig. 5, simultaneously placed two
mice in the live animal imaging system and recorded the fluorescence
3

H. Mei et al.
Journal of Photochemistry & Photobiology, A: Chemistry 418 (2021) 113335
Fig. 2. (a) Fluorescence spectral changes of titration of fluorescent probe Doc-Ac (33.33
μ
M) with Cysteine (0-2.2 equiv.) in Hepes buffer (10 mM, pH 7.3, 0.3 %
DMSO) solution system. Inset: fluorescence intensity changes of probe at 575 nm upon addition of 0-2.20 equiv. of Cys. The red line is a fitted curve (R = 0.99). (b)
UV–vis spectral changes of titration of fluorescent probe Doc-Ac (33.33
μ
M) with Cys (0-2.60 equiv.) in Hepes buffer (10 mM, pH 7.3, 0.3 % DMSO) solution. (c)
M) in the presence of various amino acids. Red bars represent the addition of a
Fluorescence intensity responses of Doc-Ac (10 M) at 575 nm toward Cys (33.33
μ
μ
single analyte (33.33 μM of each). Black bars represent the addition of each analyte together with Cys (33.33 μM). All experiments were performed in Hepes buffer
(10 mM, pH 7.3, 0.3 % DMSO) at 25 ◦C and each data was acquired 7 min after mixing. (d) Change in fluorescence intensity of probe Doc-Ac (33.33
min. after adding Cys (2.20 equiv.) in Hepes buffer (10 mM, pH 7.3, 0.3 % DMSO) solution.
μM) within 7
Scheme 2. Proposed sensing mechanism of Doc-Ac towards Cys.
distribution and fluorescence intensity at 2, 5, 7, 10, 20 and 30 min
respectively. There were an obvious fluorescence signal changes in the
subcutaneous of the mouse when the probe was injected into the mouse
body, which can be contributed to the interaction of probe with
endogenous Cys. The fluorescent signal decreased firstly and then
increased gradually and the fluorescence signal reached the weakest
values between about 7th and 10th min, and then the intensity gradually
increased a certain extent within 30 min maybe due to the metabolism
or absorption process. Another side of the mouse which was injected
with saline showed no fluorescent signal changes in the subcutaneous.
Further experiments were employed to confirm that probe Doc-Ac could
sensitively monitor Cys in vivo (Fig. 5b). In the left group was injected
subcutaneously with probe (20 M, 100 L) only, and the right group
was preinjected with excess NEM (1 mM, 100 L) to construct a mouse
model with abnormal low levels of Cys and then treated with probe (20
μ
μ
μ
μ
M, 100 μL). The mice of right group exhibited strong fluorescent signal,
whereas the left group showed no signal. Above results indicated that
the probe could be used to monitor and image endogenous Cys in
complicated living organisms.
4

H. Mei et al.
Journal of Photochemistry & Photobiology, A: Chemistry 418 (2021) 113335
Fig. 3. The 1H NMR spectra of (a) probe Doc-Ac and (b) upon addition of Cys in DMSO-d6.
Fig. 4. Fluorescence microscope images of Doc-Ac in SHSY5Y cells: (a) control; (b) SHSY5Y cells incubated with Doc-Ac (10
μ
M) for 30 min; (c) SHSY5Y cells
M) for 30 min; (d) SHSY5Y cells pretreated with NEM (1 mM) for 30 min and
then treated with Cys (100 μM) for 30 min and incubated with Doc-Ac (10 μM) for 30 min.
pretreated with NEM (1 mM) for 30 min and followed by incubation with Doc-Ac (10
μ
4. Conclusion
for the detection of Cys. The probe displayed a high selectivity and rapid
response towards Cys over other amino acids. Moreover, the probe
possessed good water-solubility, large stokes shift and low biotoxicity,
In conclusion, we have developed a novel fluorescent probe Doc-Ac
5

H. Mei et al.
Journal of Photochemistry & Photobiology, A: Chemistry 418 (2021) 113335
Fig. 5. Fluorescent detection of Cys in living mice injected with Doc-Ac in saline. (a) In vivo NIR images of living mice treated with 50
Ac (20 M, 50 L, right side) solution at different times after subcutaneous injection. (b) In vivo NIR images of the mouse preinjected intraperitoneally with or
without NEM (1 Mm) 30 min and then injected with Doc-Ac (20 M, 100 L) for 7 min.
μL of saline (left side) or Doc-
μ
μ
μ
μ
[4] R.M. Canet-Aviles, M.A. Wilson, D.W. Miller, R. Ahmad, C. Mclendon,
S. Bandyopadhyay, The Parkinson’s disease protein dj-1 is neuroprotective due to
cysteine-sulfinic acid-driven mitochondrial localization, Proc. Natl. Acad. Sci. U. S.
A. 101 (2004) 9103–9108.
which enabled it to selectively detect the intracellular Cys, except for the
faint influence of similar structure of Hcy and GSH. The results research
confirmed that Doc-Ac could be utilized as a fluorescent probe to detect
endogenous Cys in living cells and mice.
[5] E. Weerapana, C. Wang, G.M. Simon, F. Richter, S. Khare, M.B. Dillon, D.
A. Bachovchin, K. Mowen, D. Baker, B.F. Cravatt, Quantitative reactivity profiling
predicts functional cysteines in proteomes, Nature 468 (2010) 790–795.
[6] J. Xu, J. Pan, Y. Zhang, J. Liu, L. Zeng, X. Liu, Ultrasensitive near-infrared
fluorescence-enhanced probe for discriminative detection of GSH and Cys from
different emission channels, Sens. Actuators B 238 (2017) 58–65.
[7] G. Chwatko, E. Bald, Determination of cysteine in human plasma by
highperformance liquid chromatography and ultraviolet detection after precolumn
derivatization with 2-chloro-1-methylpyridinium iodide, Talanta 52 (2000)
509–515.
Author statement
Huihui Mei: Conceptualization, Methodology, Software, Investiga-
tion, Writing Original Draft.
Xin Gu and Minghui Wang: Validation, Formal analysis,
Visualization.
[8] J. Yin, Y. Hu, J. Yoon, Fluorescent probes and bioimaging: alkali metals, alkaline
earth metals and pH, Chem. Soc. Rev. 44 (2015) 4619–4644.
Yonghong Cai: Supervision, Data Curation, Writing - Review &
Editing.
[9] W. Wang, L. Li, S. Liu, C. Ma, S. Zhang, Determination of physiological thiols by
electrochemical detection with piazselenole and its application in rat breast cancer
cells 4T-1, J. Am. Chem. Soc. 130 (2008) 10846–10847.
Kuoxi Xu: Resources, Writing - Review & Editing, Supervision, Data
Curation.
[10] I. Rahman, A. Kode, S.K. Biswas, Assay for quantitative determination of
glutathione and glutathione disulfide levels using enzymatic recycling method,
Nat. Protoc. 1 (2006) 3159–3165.
Declaration of Competing Interest
[11] A.R. Ivanov, I.V. Nazimov, L.A. Baratova, Determination of biologically active low-
molecular-mass thiols in human blood. II. High-performance capillary
electrophoresis with photometric detection, J. Chromatogr. A 895 (2000) 167–171.
[12] J. Yin, Y. Kwon, D. Kim, D. Lee, G. Kim, Y. Hu, J.H. Ryu, J. Yoon, Preparation of a
cyanine based fluorescent probe for highly selective detection of glutathione and
its use in living cells and tissues of mice, Nat. Protoc. 10 (2015) 1742–1754.
[13] H.R. Zheng, L.Y. Niu, Y.Z. Chen, L.Z. Wu, C.H. Tung, Q.Z. Yang, Cascade reaction
based fluorescent probe for detection of H2S with the assistance of CTAB micelles,
Chin. Chem. Lett. 27 (2016) 1793–1796.
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Appendix A. Supplementary data
[14] Z.G. Yang, J.F. Cao, Y.X. He, J.H. Yang, T. Kim, X.J. Peng, J.S. Kim, Macro-/micro-
environment-sensitive chemosensing and biological imaging, Chem. Soc. Rev. 43
(2014) 4563–4601.
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.jphotochem.2021.
113335.
[15] F.Y. Wang, L. Zhou, C.C. Zhao, R. Wang, Q. Fei, S.H. Luo, Z.Q. Guo, H. Tian, W.
H. Zhu, A dual-response BODIPY based fluorescent probe for the discrimination of
glutathione from cystein and homocysteine, Chem. Sci. 6 (2015) 2584–2589.
[16] D.H. Ma, D. Kim, E. Seo, S.J. Lee, K. Ahn, Ratiometric fluorescence detection of
cysteine and homocysteine with a BODIPY dye by mimicking the native chemical
ligation, Analyst 140 (2015) 422–427.
References
[1] Z. Lu, Y. Lu, C. Fan, X. Sun, M. Zhang, Y. Lu, A two-separated-emission fluorescent
probe for simultaneous discrimination of Cys/Hcy and GSH upon excitation of two
different wavelengths, J. Mater. Chem. B 6 (2018) 8221–8227.
[17] X. Meng, W. Ye, S. Wang, Y. Feng, M. Chen, M. Zhu, Q. Guo, A ratiometric
twophoton fluorescent probe for cysteine and homocysteine in living cells, Sens.
Actuators B 201 (2014) 520–525.
[2] G. Liu, D. Liu, X. Han, X. Sheng, Z. Xu, S.H. Liu, L. Zeng, J. Yin, A hemicyanine-
based colorimetric and ratiometric fluorescent probe for selective detection of
cysteine and bioimaging in living cell, Talanta 170 (2017) 406–412.
[18] C. Xu, H. Li, B. Yin, A colorimetric and ratiometric fluorescent probe for selective
detection and cellular imaging of glutathione, Biosens. Bioelectron. 72 (2015)
275–281.
[3] C.E. Paulsen, K.S. Carroll, Cysteine-mediated redox signaling: chemistry, biology,
and tools for discovery, Chem. Rev. 113 (2013) 4633–4679.
6

H. Mei et al.
Journal of Photochemistry & Photobiology, A: Chemistry 418 (2021) 113335
[19] H. Chen, Y. Tang, M. Ren, W. Lin, Single near-infrared fluorescent probe with
highand low-sensitivity sites for sensing different concentration ranges of
biological thiols with distinct modes of fluorescence signals, Chem. Sci. 7 (2016)
1896–1903.
[33] Q. Ye, S.S. Chen, D.D. Zhu, X.M. Lu, Q.H. Lu, Preparation of aggregation-induced
emission dots for long-term two-photon cell imaging, J. Mater. Chem. B 3 (2015)
3091–3097.
[34] J.K. Jaiswal, H. Mattoussi, J.M. Mauro, S.M. Simon, Long-term multiple color
imaging of live cells using quantum dot bioconjugates, Nat. Biotechnol. 21 (2002)
47–51.
[20] X. Dai, T. Zhang, J. Miao, B. Zhao, A ratiometric fluorescent probe with DNBS
group for biothiols in aqueous solution, Sens. Actuators B 223 (2016) 274–279.
[21] A.R. Sarkar, C.H. Heo, E.J. Kim, H.W. Lee, H. Singh, J.J. Kim, H. Kang, C. Kang, H.
M. Kim, A cysteamine-selective two-photon fluorescent probe for ratiometric
bioimaging, Chem. Commun. 51 (2015) 2407–2410.
[35] A.R. Lippert, G.C. Van De Bittner, C.J. Chang, L. Zhang, Y. Zhang, Y. Du, N.S.
K. Gowthaman, P. Arul, J.J. Shim, K. Kenry, K.C. Chong, B. Liu, Boronate oxidation
as a bioorthogonal reaction approach for studying the chemistry of hydrogen
peroxide in living systems, Acc. Chem. Res. 44 (2011) 793–804.
[36] Y.K. Yue, F.J. Huo, C.X. Yin, J.O. Escobedo, R.M. Strongin, Recent progress in
chromogenic and fluorogenic chemosensors for hypochlorous acid, Analyst 141
(2016) 1859–1873.
[22] Y. Qi, Huang, B. Li, F. Zeng, S.Z. Wu, Real-time monitoring of endogenous cysteine
levels in vivo by near-infrared turn-on fluorescent probe with large stokes shift,
Anal. Chem. 90 (2018) 1014–1020.
[23] X. Gao, X. Li, L. Li, J. Zhou, H. Ma, A simple fluorescent off-on probe for the
discrimination of cysteine from glutathione, Chem. Commun. 51 (2015)
9388–9390.
[37] D.H. Yu, Q.S. Zhai, S.J. Yang, G.Q. Feng, A colorimetric and near-infrared
fluorescent turn-on probe for in vitro and in vivo detection of thiophenols, Anal.
Methods 7 (2015) 7534–7539.
[24] F. Wang, Z. Guo, X. Li, X. Li, C. Zhao, Development of a small molecule probe
capable of discriminating cysteine, homocysteine, and glutathione with three
distinct turn-on fluorescent outputs, Chem. Eur. J. 20 (2014) 11471–11478.
[25] Y. Yang, F. Huo, C. Yin, J. Chao, Y. Zhang, An ‘OFF-ON’ fluorescent probe for
specially recognize on Cys and its application in bioimaging, Dyes Pigm. 114
(2015) 105–109.
[38] P. Wang, J. Fu, K. Yao, Y. Chang, K. Xu, Y. Xu, A novel quinoline-derived
fluorescent “turn-on” probe for Cu2+ with highly selectivity and sensitivity and its
application in cell imaging, Sens. Actuators B 273 (2018) 1070–1076.
[39] Y. Chang, H. Qin, X. Wang, X. Li, M. Li, H. Yang, K. Xu, G. Qing, Visible and
reversible restrict of molecular configuration by copper ion and pyrophosphate,
ACS Sens. 5 (2020) 2438–2447.
[26] R. Chand, S.K. Jha, K. Islam, D. Han, I.S. Shin, Y.S. Kim, Analytical detection of
biological thiols in a microchip capillary channel, Biosens. Bioelectron. 40 (2013)
362–367.
[40] Y. Dai, K. Yao, J. Fu, K. Xue, L. Yang, K. Xu, A novel 2-(Hydroxymethyl)quinolin-8-
ol-based selective and sensitive fluorescence probe for Cd2+ ion in water and living
cells, Sens. Actuators B 251 (2017) 877–884.
[27] Y. Liu, D. Yu, S. Ding, Q. Xiao, J. Guo, G. Feng, Rapid and ratiometric fluorescent
detection of cysteine with high selectivity and sensitivity by a simple and readily
available probe, ACS Appl. Mater. Interfaces 6 (2014) 17543–17550.
[28] H. Li, L. Jin, Y. Kan, B. Yin, A visual and “turn-on” fluorescent probe for rapid
detection of cysteine over homocysteine and glutathione, Sens. Actuators B 196
(2014) 546–554.
[41] Y. Chang, J. Fu, K. Yao, B. Li, K. Xu, X. Pang, Novel fluorescent probes for
sequential detection of Cu2+ and citrate anion and application in living cell
imaging, Dyes Pigm. 161 (2019) 331–340.
[42] J. Fu, Y. Chang, B. Li, H. Mei, K. Xu, An aminoquinoline based fluorescent probe for
sequential detection of Znic (II) and inorganic phosphate and application in living
cell imaging, Appl. Organometal. Chem. 33 (2019) e5162.
[29] C. Chen, L. Zhou, X. Huang, W. Liu, Rapid detection of intracellular Cys over Hcy
and GSH using a novel two-photon coumarinocoumarin-based colorimetric and
fluorescent probe, J. Mater. Chem. B 5 (2017) 58923–58927.
[43] B. Li, J. Kou, H. Mei, X. Gu, M. Wang, X. Xie, K. Xu, A hemicyanine-based “turn-on”
fluorescent probe for the selective detection of Cu2+ ions and imaging in living
cells, Anal. Methods 12 (2020) 4181–4184.
[30] X. Zhang, L. Zhang, W.W. Ma, Y. Zhou, Z.N. Lu, S. Xu, A near-infrared ratiometric
fluorescent probe for highly selective recognition and bioimaging of cysteine,
Front. Chem. 7 (2019) 32.
[44] K.X. Xu, X.M. Xie, H.J. Kong, P. Li, J.L. Zhang, X.B. Pang, Selective fluorescent
sensors for malate anion using the complex of phenanthroline-based Eu(III) in
aqueous solution, Sens. Actuators B 201 (2014) 131–137.
[31] Y. Long, J. Liu, D. Tian, F. Dai, S. Zhang, B. Zhou, Cooperation of ESIPT and ICT
processes in the designed 2-(2′-hydroxyphenyl)benzothiazole derivative: a near-
infrared two-photon fluorescent probe with a large stokes shift for the detection of
cysteine and its application in biological environments, Anal. Chem. 92 (2020)
14236–14243.
[45] Y. Dai, K. Xu, Q. Li, C. Wang, X. Liu, P. Wang, Acridine-based complex as amino
acid anion fluorescent sensor in aqueous solution, Spectrochim. Acta A 157 (2016)
1–5.
[46] G.L. Long, J.D. Winefordner, Limit of detection. A closer look at the IUPAC
definition, Anal. Chem. 55 (1983), 712A–24A.
[32] X.M. Wu, X.R. Sun, Z.Q. Guo, J.B. Tang, Y.Q. Shen, T.D. James, H. Tian, W.H. Zhu,
In vivo and in situ tracking cancer chemotherapy by highly photostable NIR
fluorescent theranostic prodrug, J. Am. Chem. Soc. 136 (2014) 3579–3588.
[47] X. Luo, X. Li, Y. Wang, X. Xie, L. Yang, A novel reversible fluorescent probe for
sequential detection of aluminium ion and PPi and bioimaging in living cells,
ChemistrySelect 4 (2019) 10643–10648.
7